Significance of Caspase-Mediated Cleavage of PARP-1 in Cell Death Poly (ADP-ribose) polymerase-1 (PARP-1) maintains cell function by facilitating DNA repair. However, under pathological conditions, PARP-1 mediates cell death in experimental models of ischemia- reperfusion injury, myocardial infarction, glutamate excitotoxicity, and Parkinson disease. Thus, PARP-1 inhibition has been a therapeutic focus for aging-related diseases such as stroke, heart attack and neurodegeneration. PARP-1 facilitates DNA repair by producing poly(ADP-ribose) polymers (PAR) using NAD+ as a substrate, and attaching PAR to itself and other proteins. With excessive DNA damage however, PARP-1 overactivation leads to cell demise through depletion of NAD+ and overproduction of PAR. As such, understanding how PARP-1's activity is regulated during cell death will be valuable for halting aging and disease progression. One possible form of PARP-1 regulation during apoptotic cell death occurs when active caspases cleave PARP-1 into 89 and 24 kDa fragments possibly leading to PARP-1 inactivation. This cleavage has long been regarded as a biochemical hallmark of apoptosis, but how this cleavage affects PARP-1's physiological and pathological roles remains unclear. Since PARP-1's enzymatic function consumes NAD+, it is believed that this cleavage prevents PARP-1 over-activation from depleting cellular energy. Alternatively, in cell death pathways where PARP-1 promotes survival through DNA repair, PARP-1 cleavage may be necessary for the progression of cell death. To directly investigate the role of PARP- 1 cleavage in cell death, we generated a PARP-1 knock-in (PKI) mouse in which the caspase cleavage site of PARP-1 was mutated. We have confirmed that the mutation renders PARP-1 uncleavable by caspases in both neuronal and fibroblast cultures. How PARP-1 activity is affected by caspase cleavage will depend on the cell type studied and the death stimuli used. Thus, to delineate the cell death pathways in which caspase cleavage of PARP-1 are important, we will compare susceptibility of WT and PKI fibroblasts and neurons to a variety of cellular toxins. In addition, to determine the effect of PARP-1 cleavage in excitotoxic cell death, we will subject mice to experimental models of ischemic injury in which the brain injury is primarily mediated by PARP-1 over-activation. These studies will help to identify the functional significance of PARP-1 cleavage in different paradigms of cell death. With PARP-1's diverse roles in cellular homeostasis, aging, and disease, understanding how its activity is regulated may identify points of intervention for halting aging and disease progression. PARP-1 is cleaved by caspases during cell death, and this phenomenon has long been regarded as a biochemical hallmark for apoptosis. We believe that understanding the functional significance of this cleavage will help in developing therapeutic intervention to combat aging-related diseases and cell death. [unreadable] [unreadable] [unreadable]